Saccharomyces Cerevisiae Strains that Overexpress Heterologous Proteins

Sleep, Darrell; Belfield, Graham; Ballance, D. J.; Steven, John; Jones, S. D. M.; Evans, Leslie; Moir, Peter D.; Goodey, A. R.

doi:10.1038/nbt0291-183

Cited by 59 publications

(40 citation statements)

References 35 publications

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“…Thus, the development of an alternative method of industrial preparation of HSA is desirable. The development of genetic engineering has opened up the possibility of producing recombinant human serum albumin (rHSA) without the danger of contamination by human pathogens, and at lower cost (Saunders et al, 1987, Sleep et al, 1991, Fleer et al, 1991, Hodgkins et al, 1990, Wartmann et al, 2002.…”

Section: Pagementioning

confidence: 99%

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Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Zhang¹,

Li²,

Agblevor³

2005

Process Biochemistry

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The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (k L a) and subsequently increased the cell mass and protein production compared to conventional fermentation.Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in the conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. The volumetric oxygen transfer coefficient k L a was 1011.9 h -1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h -1 ) but was similar to the conventional 750 rpm (1098 h -1 ). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging.Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. ACKNOWLEDGMENTS

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Section: Pagementioning

confidence: 99%

“…Saccharomyces cerevisiae strains have long been used as an expression system to secrete rHSA (Sleep et al, 1991). Stable multicopy vectors were also designed for efficient secretion of rHSA by industrial strains of Kluyveromyces yeasts (Fleer et al, 1991).…”

Section: Human Serum Albuminmentioning

confidence: 99%

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Zhang¹,

Li²,

Agblevor³

2005

Process Biochemistry

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“…The three N-terminal PD-ECGF variant sequences were inserted into a pJDB207-based yeast episomal expression plasmid. Transcription initiation and termination sequences were provided by the S. cerevisiae PRBl promoter and ADHl terminator sequences (Sleep et al, 1991).…”

Section: Expression Of Recombinant Pd-ecgfmentioning

confidence: 99%

Thymidine phosphorylase activity of platelet‐derived endothelial cell growth factor is responsible for endothelial cell mitogenicity

Finnis

Dodsworth

Pollitt

et al. 1993

European Journal of Biochemistry

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Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98 % purity by anion-exchange and hydroxyapatite chromatography. It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; K,, 0.11 mM; V, , , 12.5 mmol min-' mg-'; turnover number, 9.4 s -I ) . Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

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“…The GFUpA-PAI-2 cDNA was inserted in a pJDB207-based [26] yeast expression vector under the transcriptional control of the S. cerevisiae protease B (PRBI) gene promoter [17]. The plasmid also contains the LEU2d gene as a selectable marker and part of the 2-pm plasmid to provide replication functions.…”

Section: Expression Of Gffupa -Pai-2 In S Cerevisiaementioning

confidence: 99%

“…The Hind111 fragment containing the synthesised hybrid gene was then inserted into an expression vector based on the S. ccrcvisiar PRBI promoter [17] to form pDJBl39. In addition to the PRBI promoter and the coding sequence, this plasmid contains the A D H f terminator, the LEU2d gene and part of the 2-pm plasmid.…”

Section: Nbldlllmentioning

confidence: 99%

A hybrid protein of urokinase growth‐factor domain and plasminogen‐activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor

Ballance

Marshall

Cottingham

et al. 1992

European Journal of Biochemistry

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The binding of urokinase‐type plasminogen activator (uPA) to its specific cell‐surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR‐binding growth‐factor domain of uPA (amino acids 1–47; GFuPA) at the N‐terminus of plasminogen‐activator inhibitor type 2 (PAI‐2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI‐2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA–PAI‐2 competed with uPA, the N‐terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4–43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA‐inhibitory and uPAR‐binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine‐protease‐inhibitor superfamily.

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Saccharomyces Cerevisiae Strains that Overexpress Heterologous Proteins

Cited by 59 publications

References 35 publications

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Thymidine phosphorylase activity of platelet‐derived endothelial cell growth factor is responsible for endothelial cell mitogenicity

A hybrid protein of urokinase growth‐factor domain and plasminogen‐activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor

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